Download Inhaled heparin in cystic fibrosis D.J. Serisier* , J.K. Shute , P.M. Hockey

Eur Respir J 2006; 27: 354–358
DOI: 10.1183/09031936.06.00069005
CopyrightßERS Journals Ltd 2006
Inhaled heparin in cystic fibrosis
D.J. Serisier*,#, J.K. Shute", P.M. Hockey#, B. Higgins+, J. Conway1 and M.P. Carroll#
ABSTRACT: Cystic fibrosis (CF) is characterised by inspissated airway secretions and chronic
endobronchial infection associated with exuberant neutrophilic inflammation. Unfractionated
heparin may be mucolytic and has demonstrated a number of anti-inflammatory properties;
however, further safety data are needed in these subjects who are at risk of airway bleeding. The
current study aimed to assess the medium-term safety and tolerability of moderately high-dose
inhaled heparin in CF adults and to explore possible in vivo mucolytic and anti-inflammatory
outcomes.
A randomised, double-blind, placebo-controlled crossover study of twice daily inhalation of
50,000 IU of heparin for 2 weeks was undertaken in CF adults, with a 1-week washout period.
Eighteen subjects were randomised and 14 (mean¡SD age 23¡7.8 yrs and percentage-predicted
forced expiratory volume in one second 52.1¡15.56%) completed the study protocol.
Heparin neither affected blood coagulation parameters nor resulted in any increase in adverse
events. Heparin inhalation had no significant effect upon forced expiratory volume in one second,
symptoms of sputum clearance or sputum inflammatory markers.
The current pilot study demonstrated no evidence of improved sputum clearance with 50,000 IU
of inhaled heparin given twice daily to adult cystic fibrosis subjects. However, inhaled heparin
was safe and the future evaluation of larger doses over a longer period may be warranted.
KEYWORDS: Cystic fibrosis, heparin, inflammation, mucolytic, neutrophil elastase
nspissated respiratory secretions and chronic
airway inflammation are central to the
progressive pulmonary damage that characterises cystic fibrosis (CF), ultimately culminating
in respiratory failure and death. Intense neutrophilic activity is central to the inflammatory
process [1], augmented by excessive levels of
proinflammatory cytokines [2], particularly interleukin (IL)-8 [3], and the destructive inflammatory serine protease, neutrophil elastase [4].
I
The systemic anti-inflammatory therapies prednisone and ibuprofen both improve lung function
in CF [5, 6]; however, neither appears to be
effective in adults [5, 7] and both have significant
potential side-effects. Macrolide antibiotics have
shown interesting anti-inflammatory properties,
although the mechanism of their clinical efficacy
in CF subjects remains unclear [8].
Enhanced clearance of retained secretions using
recombinant human deoxyribonuclease (DNase)
also improves lung function in CF [9]. Furthermore, improved clearance of respiratory secretions may reduce airway inflammation, as DNase
appears to attenuate the progressive increase in
airway inflammation that occurs in CF [10].
However, DNase is expensive, and a substantial
proportion of CF subjects do not appear to derive
benefit [11].
354
VOLUME 27 NUMBER 2
There are reports of increased sputum expectoration with unfractionated heparin [12, 13], and
charged oligosaccharides have shown mucolytic
effects in vitro [14], suggesting that heparin may
enhance airway clearance. Furthermore, heparin
has displayed a diverse range of in vitro antiinflammatory and immunomodulatory effects,
including inhibitory effects upon leukocyte
recruitment [15], neutrophil chemotaxis [16],
neutrophil elastase [17] and chemokines, such
as IL-8 [18]. Excess neutrophil elastase activity
plays a key role in contributing to airway
obstruction in CF by multiple mechanisms,
including stimulating IL-8 production [19] and
mucus secretion [20], impairing ciliary function
[21], and potentially desiccating airway secretions by activating epithelial sodium channels
[22]. Recently, the current authors observed
significant reductions in sputum neutrophil
elastase activity ex vivo with increasing doses of
inhaled heparin in a small group of CF subjects
[23], with several subjects commenting upon
immediate enhancement of sputum clearance
following the higher doses.
Despite the recent demonstration of the safety of
single high doses of inhaled heparin in normal
subjects [24], CF subjects have specific safety
issues related to airway bleeding. The current
study aimed to assess the medium-term safety
AFFILIATIONS
*Dept of Respiratory Medicine, Mater
Adult Hospital, Brisbane, Australia.
#
Adult Cystic Fibrosis Unit,
Southampton General Hospitals NHS
Trust, and
1
Respiratory Cell and Molecular
Biology Division, Southampton
University, Southampton,
"
School of Pharmacy and Biomedical
Sciences, and
+
Dept of Mathematics, University of
Portsmouth, Portsmouth, UK.
CORRESPONDENCE
D.J. Serisier
Dept of Respiratory Medicine
Mater Adult Hospital
Brisbane, 4101
Australia
Fax: 61 738402402
E-mail: [email protected]
Received:
June 13 2005
Accepted after revision:
October 03 2005
European Respiratory Journal
Print ISSN 0903-1936
Online ISSN 1399-3003
EUROPEAN RESPIRATORY JOURNAL
D.J. SERISIER ET AL.
and tolerability of moderately high dose inhaled unfractionated heparin in CF adults and to explore possible in vivo
mucolytic (assessed by lung function, symptom scores and
quality of life) and anti-inflammatory outcomes. The study was
powered to detect a 10% improvement in forced expiratory
volume in one second (FEV1) as a result of enhanced sputum
clearance, consistent with the short-term effect demonstrated
in early studies of DNase [25].
METHODS
Subjects
Nonsmoking adults with CF were recruited from the adult CF
clinic at Southampton General Hospital, Southampton, UK, if
they had been clinically stable for 21 days. The protocol was
approved by the hospital ethics committee and all subjects
provided written informed consent. At enrolment, FEV1 was
required to be within 10% of the best recorded value in the
preceding 6 months.
Exclusion criteria were pregnancy, allergy to heparins, or any
of the following within the preceding 21 days: symptoms of
infective exacerbation; prescription of supplemental antibiotic
therapy; and change in maintenance therapies or haemoptysis.
Subjects with a bleeding diathesis, including platelet count
,1006106 cells?mL-1, or international normalised ratio or
activated partial thromboplastin ratio .1.25, were also
excluded.
Study design and procedures
This was a randomised, double-blind, placebo-controlled
crossover study. Subjects were allocated in random order to
consecutive 2-week treatments of inhaled, twice daily, 4-mL
0.9% saline or 50,000 IU of heparin sodium in injectable form
(25,000 IU?mL-1; Leo Laboratories, Princes Risborough, UK;
made up to 4 mL with 0.9% sodium chloride solution). All
study treatments were administered with a Sidestream jet
nebuliser and Portaneb compressor (Medic-Aid, Bognor Regis,
UK), after taking short-acting b-agonist and performing usual
chest physiotherapy. All usual medications were maintained
throughout the entire trial period. Subjects were directed to
return unused doses at the completion of each of the
treatments to enable an estimation of treatment compliance.
Randomisation was performed using computer-generated
block randomisation in groups of four, supplied by the
Statistics Dept and held by the Pharmacy Dept. The
Pharmacy Dept allocated, prepared and supplied trial drug
to sequential subjects according to their randomisation code.
Trial subjects, trial supervisors and all staff involved in direct
patient care were unaware of treatment allocations at all times.
Code-breaking and allocation sheets for emergency use were
kept by the Pharmacy Dept, but were never used. Heparin and
saline were provided as identical, clear, colourless solutions.
INHALED HEPARIN IN CYSTIC FIBROSIS
administration of 14 days of antibiotic therapy, followed by a
further 21-day period of clinical stability prior to commencing
treatment two. This maximised the equivalence of subjects for
the two treatments.
Withdrawal criteria included haemoptysis o20 mL, pulmonary infective exacerbation or any requirement for antibiotic
therapy during treatment periods. However, heparin inhalation has been associated with increased sputum expectoration
[12, 13], which may result in subjects interpreting a decline in
their respiratory status. In an attempt to minimise the possible
influence of such a subjective effect upon trial outcomes (and
given that there is no universally agreed definition for an
infective exacerbation), infective exacerbation was defined as a
requirement for oral or intravenous antibiotic therapy associated with a 10% or greater decline in FEV1 and increase in
serum C-reactive protein (CRP; of at least 10 mg?L-1 and at
least 20% from baseline), in addition to at least two of the
following: increased sputum purulence; increased sputum
volume; new or increased haemoptysis; fevers or systemic
symptoms; weight loss of at least 1 kg; new crackles on chest
auscultation; and new infiltrates on chest radiograph.
Safety was assessed by both blood coagulation indices and
incidence of adverse events. The primary outcome measure
was change in FEV1, comparing heparin and placebo treatments. Secondary outcome measures were changes in the
following: sputum levels of neutrophil elastase activity, IL-8,
terminal complement complex (TCC) and myeloperoxidase
(MPO); serum CRP; weight; symptom scores; quality of life
and exercise capacity.
Spirometry was performed according to American Thoracic
Society criteria [26], using a Sensormedics rolling seal
spirometer (Sensormedics; Viasys Healthcare, Yorba Linda,
CA, USA). Timing of spirometry was kept consistent to within
2 h during the trial. To assess exercise tolerance, patients
underwent a maximal, incremental 10-m shuttle walking test,
as previously described [27]. Visual analogue scores for eight
symptoms (general well-being, energy, appetite, sleep quality,
shortness of breath, cough, sputum volume and sputum
colour) were completed at each visit without reference to
prior scores or answers. Subjects also completed the teen–
adult version of the Cystic Fibrosis Questionnaire (CFQ),
a validated, disease-specific, health-related quality-of-life
measure [28].
The dose of heparin sodium selected was based upon the
current authors’ preceding pilot study [23], which showed
significant reductions in sputum neutrophil elastase activity
following single-inhaled doses of both 1,000 and 2,000 IU?kg-1.
There was a 1-week washout period between treatments. In the
event that any subject’s FEV1 at the start of the second
treatment was ,90% of their baseline FEV1 at commencement
of treatment one, the washout period was prolonged to allow
At each study visit, spontaneously expectorated sputum
samples were collected in airtight sterile containers and stored
at -80uC for subsequent processing. After thawing samples on
ice, salivary contamination was removed by pipette and the
sample diluted (1:1 mL?g-1) with PBS (Gibco, Paisley, UK) for
solubilisation of mediators. The samples were thoroughly
vortexed on a whirly mixer for 5 min, mixed on an orbital plate
mixer for 20 min on ice, then centrifuged at 20,0006g, at 4uC
for 20 min. The supernatant was aspirated and used to
determine neutrophil elastase activity, IL-8, MPO and TCC
levels. Commercially available ELISA kits were used to
measure total IL-8 (Pelikine compact; Mast Diagnostics,
Liverpool, UK) and TCC (Quidel, San Diego, CA, USA).
MPO was measured using a commercially available RIA kit
(Pharmacia, Uppsala, Sweden). Neutrophil elastase activity
EUROPEAN RESPIRATORY JOURNAL
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INHALED HEPARIN IN CYSTIC FIBROSIS
D.J. SERISIER ET AL.
was measured in samples diluted 1:10 with assay buffer, as
previously described [29].
Statistical analysis
The current authors’ sample size calculation indicated the need
for a minimum of 13 evaluable patients to detect a difference in
change in FEV1 of 0.14 L (equivalent to 10% difference in mean
scores), with 95% power at the 5% (two-sided) level of
significance. This assumed a SD of 0.123 L for the difference
between paired observations estimated from the current
authors’ pilot data [22].
The sample size was increased to 18 subjects (a 30% increase)
to allow for attrition. To be eligible for full outcome analysis, a
patient had to be recruited, randomised, and had to complete
both arms of the trial without dropout; however, safety data
were collected for all subjects. All measurements, data
collection and data entry were completed before treatment
codes were broken.
Analyses compared within-subject differences for changes in
outcomes between heparin and placebo. For all such differences in change that were normally distributed, a complete
crossover ANOVA for repeated measures was performed,
including assessment for period effects and treatment–period
interaction. If there was evidence of period effect or treatment–
period interaction, covariate analyses were performed upon
treatment period one data alone, treating the data as per a
parallel study. The Wilcoxon signed-rank test was employed
for non-normally distributed outcome data. Comparison of
groups for determination of differences in the incidence of
side-effects and baseline variables used McNemar’s test for
analysis of paired proportions. Pearson’s product moment
correlation coefficients were computed to assess the degree of
association between numeric variables on appropriate measurement scales. p-Values ,0.05 were considered significant.
RESULTS
Eighteen subjects were randomised and four were subsequently withdrawn, two in relation to heparin treatment (both
infective exacerbation) and two in relation to placebo (one for
noncompliance and one for personal reasons). The 14 remaining subjects completed both treatment periods; eight received
heparin first and six received placebo first. Table 1 shows the
patient characteristics. There were no differences in the clinical
characteristics of the subjects at the start of the heparin and
placebo periods. Four subjects had their washout periods
prolonged; three received oral antibiotics and one received
intravenous antibiotics.
The average compliance was 95% for heparin and 96% for
placebo. There were no significant adverse events attributable
to heparin. The incidence of side-effects did not differ between
heparin and placebo. No subject experienced haemoptysis
whilst inhaling heparin. No subject discontinued the trial due
to treatment intolerance on either arm. Heparin had no effect
upon blood coagulation indices, including platelet count
(treatment effect -7.16106 cells?mL-1; p50.67; 95% confidence
interval (CI) -42.7–28.5), activated partial thromboplastin time
or international normalised ratio. Infective exacerbations
developed in eight of the 18 randomised subjects during the
study, three during treatment and five during washout. Five
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VOLUME 27 NUMBER 2
TABLE 1
Baseline characteristics of the study population
Average
Age yrs
23.8¡7.8
Female n
10
FEV1 L
1.71¡0.52
FEV1 %
52.7¡16
Weight kg
55.3¡8.4
Body mass index kg?m-2
20.5¡2
Microbiology n#
Pseudomonas aeruginosa
11
Burkholderia cepacia
1
Staphylococcus aureus
3
Medications n
Bronchodilators
9
Inhaled corticosteroids
11
Inhaled antibiotics
12
Inhaled deoxyribonuclease
1
Azithromycin
3
Data are presented as mean¡SD, unless otherwise indicated. FEV1: forced
expiratory volume in one second; FEV1 %: FEV1 as a percentage of the
predicted value.
#
: subjects with chronic pulmonary colonisation by the
indicated organism (some subjects were colonised with .1 organism). n514.
occurred in relation to heparin treatment and three in relation
to placebo (p50.73).
There were no significant differences between the heparin and
placebo arms for incidence of cough (p50.50) or unusual taste
(p50.63) with inhalation of trial drug. Visual analogue scores
for cough did not differ significantly between heparin and
placebo (treatment effect -0.08; p50.87; 95% CI -1.11–0.95).
Outcomes
There was no significant effect of heparin upon the primary
outcome measure, FEV1 (treatment effect -0.008 L; p50.90; 95%
CI -0.15–0.13; table 2), or the forced vital capacity or inspiratory
capacity. There was a trend to treatment by period interaction
for FEV1 (p50.061); however, covariate analysis performed on
treatment period one did not alter any results (mean difference
-0.08 L; p50.36; 95% CI -0.26–0.1; table 2). Heparin inhalation
was associated with individual improvements in FEV1 of o10%
in two subjects and a decline of o10% in one.
There was no significant effect of heparin upon visual
analogue scores (including those pertaining to sputum
clearance; table 2), overall quality of life on the CFQ, or any
of the three quality-of-life modules. Heparin was associated
with negative effects upon both the body image (median
difference -5.56; p50.04) and social/marginalisation dimensions (median difference -6.67; p50.07); however, these were
not significant after incorporating a multiple comparisons
adjustment for the 15 quality-of-life measures of the CFQ (after
a Bonferroni adjustment, significance at the 5% level is
indicated by a p-value ,0.003).
There were no significant effects of heparin inhalation upon
weight, serum CRP or exercise tolerance. Inhaled heparin had
no significant effect upon any of the sputum inflammatory
markers (table 2).
EUROPEAN RESPIRATORY JOURNAL
D.J. SERISIER ET AL.
TABLE 2
INHALED HEPARIN IN CYSTIC FIBROSIS
Changes in selected outcome measures according to treatment regimen
Heparin
Placebo
Treatment effect#
p-value
Lung functione
FEV1 L
-0.01 (-0.08–0.06)
-0.004 (-0.074–0.066)
-0.008 (-0.15–0.13)
0.90"
FEV1 L
-0.03 (-0.13–0.07)
0.05 (-0.04–0.14)
-0.08 (-0.26–0.1)
0.36+
Sputum volume
0.28 (-0.78–1.34)
-0.01 (-0.76–0.74)
0.22 (-1.26–1.71)
0.75"
Sputum purulence
0.31 (-0.34–0.96)
-0.19 (-0.87–0.49)
0.50 (-0.65–1.66)
0.36"
1.3 (-8.9–11.5)
-3.3 (-8.7–2.1)
5.55 (-7.3–18.3)
0.36"
Sputum symptom scores
Sputum inflammatory markers##
NE activity mU?mL-1
2
-1
0.19 (-27.3–48.9)
-0.12 (-4.9–8.4)
0.30 (-35.7–53.8)
0.701
MPO mg?L-1
15.9 (-29.86–61.66)
-12.5 (-44–19)
32.72 (-35.2–100.7)
0.31#
TCC ng?mL-1
16.85 (-55.45–89.15)
-19.4 (-59.7–20.9)
-2.85 (-38.8–33.1)
0.87#
Total IL-8 610 ng?mL
Data are presented as mean (95% confidence interval), except interleukin (IL)-8, which is presented as median (range). FEV1: forced expiratory volume in one second;
NE: neutrophil elastase; MPO: myeloperoxidase; TCC: terminal complement complex. #: Treatment effect represents the difference between heparin and placebo, in
mean (median) values for normal (non-normal) data; ": full crossover ANOVA analysis; +: covariate analysis on treatment period one only; 1: Wilcoxon signed-rank test;
e
: n514 for FEV1;
##
n513 for sputum markers.
DISCUSSION
The current study investigates both the largest dose and
longest duration of inhaled heparin yet reported in any clinical
trial of subjects with lung disease. Despite evaluating a group
of patients potentially at risk of pulmonary bleeding, inhaled
heparin was safe and well tolerated, with no bleeding
complications. Furthermore, consistent with the short half-life
of heparin and data demonstrating little systemic absorption
from the tracheobronchial tree, there was no evidence of any
cumulative effect of heparin upon systemic coagulation
markers, extending previous findings [12, 24, 30, 31].
This trial failed to detect any clinical benefit of 2 weeks of
heparin inhalation in chronically productive adult CF subjects
with moderate lung disease. The study was powered to detect
a large treatment effect of inhaled heparin on FEV1 and the
treatment duration was necessarily limited for safety reasons.
Therefore, this study was not designed or powered adequately
to exclude an effect of heparin upon clinical measures,
including lung function. Although no evidence of enhanced
sputum clearance (on the basis of symptom scores, quality of
life or lung function) was found, recent work within the
current authors’ laboratory provides further evidence of
mucolytic activity of unfractionated heparin in vitro
(unpublished data), and a longer treatment duration or larger
dose may therefore be needed to result in mucolysis in vivo.
may have resulted in spurious findings in those prior
uncontrolled studies, and further dose-ranging studies are
needed.
In conclusion, inhaled heparin at a dose of 50,000 IU
administered twice daily for 2 weeks was well tolerated in
cystic fibrosis adults and did not result in any bleeding
complications or evidence of adverse events. This provides
support for its future cautious evaluation in larger doses, for
longer periods, or in subjects with early lung disease.
The inability to demonstrate any ex vivo anti-inflammatory
effect of heparin contrasts with prior demonstrations of rapid
reductions in neutrophil elastase activity [23] and IL-8 and IL-6
[12] in sputum. It is possible that this is the result of insufficient
dosing or inadequate duration, although reductions in sputum
inflammatory markers were previously shown with only
25,000 IU of inhaled heparin given once daily for 7 days [12]
and with single doses of both of the higher doses (1,000 and
2,000 IU?kg-1) in the current authors’ pilot study [23]. The
substantial variability in measures of inflammatory mediators
in spontaneously expectorated sputum from CF subjects [32]
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